U.S. patent application number 16/327835 was filed with the patent office on 2019-06-27 for modular specimen holders for high pressure freezing and x-ray crystallography of a specimen.
The applicant listed for this patent is LEICA MIKROSYSTEME GMBH. Invention is credited to Heinz PLANK, Siegfried TANKI, Cveta TOMOVA, Rainer WOGRITSCH, Paul WURZINGER.
Application Number | 20190195814 16/327835 |
Document ID | / |
Family ID | 56883557 |
Filed Date | 2019-06-27 |
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United States Patent
Application |
20190195814 |
Kind Code |
A1 |
WOGRITSCH; Rainer ; et
al. |
June 27, 2019 |
MODULAR SPECIMEN HOLDERS FOR HIGH PRESSURE FREEZING AND X-RAY
CRYSTALLOGRAPHY OF A SPECIMEN
Abstract
The present invention relates to modular specimen holder (10')
for high pressure freezing and/or X-ray crystallography of a
specimen comprising a specimen holding element (100') and an
extension element (200') connectable with each other and separable
from each other; the specimen holding element (100') comprising a
tubule (120') and a base element (110'), wherein the tubule (120')
is adapted to hold the specimen, the base element (110') is adapted
to hold the tubule (120'), wherein a distance from a bottom of the
base element (110') to a top of the tubule (120') is a distance
d.sub.1; the extension element (200') being adapted to be connected
with the base element (110'), wherein, when the extension element
(200') and the base element (110') are connected with each other, a
distance from a bottom of the extension element (200') to the top
of the tubule (120') is a second distance d.sub.2; wherein the
second distance d.sub.2 is larger than the first distance
d.sub.1.
Inventors: |
WOGRITSCH; Rainer; (Vienna,
AT) ; TOMOVA; Cveta; (Vienna, AT) ; WURZINGER;
Paul; (Deutsch-Wagram, AT) ; PLANK; Heinz;
(Wiener Neudorf, AT) ; TANKI; Siegfried; (Vienna,
AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LEICA MIKROSYSTEME GMBH |
Vienna |
|
AT |
|
|
Family ID: |
56883557 |
Appl. No.: |
16/327835 |
Filed: |
July 13, 2017 |
PCT Filed: |
July 13, 2017 |
PCT NO: |
PCT/EP2017/067696 |
371 Date: |
February 24, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 1/42 20130101; G01N
23/20033 20130101; G01N 23/20025 20130101; G01N 23/20041
20130101 |
International
Class: |
G01N 23/20033 20060101
G01N023/20033; G01N 1/42 20060101 G01N001/42 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 26, 2016 |
EP |
16186050.7 |
Claims
1. A modular specimen holder (10, 10') for high pressure freezing
and/or X-ray crystallography of a specimen comprising a specimen
holding element (100, 100') and an extension element (200, 200')
connectable with each other and separable from each other; the
specimen holding element (100, 100') comprising a tubule (120,
120') and a base element (110, 110'), wherein: the tubule (120,
120') is adapted to hold the specimen, the base element (110, 110')
is adapted to hold the tubule (120, 120'), a distance from a bottom
of the base element (110, 110') to a top of the tubule (120, 120')
is a distance d.sub.1; the extension element (200, 200') being
adapted to be connected with the base element (110, 110'), wherein,
when the extension element (200, 200') and the base element (110,
110') are connected with each other, a distance from a bottom of
the extension element (200, 200') to the top of the tubule (120,
120') is a second distance d.sub.2; wherein the second distance
d.sub.2 is larger than the first distance d.sub.1.
2. The modular specimen holder (10, 10') according to claim 1,
wherein the first distance d.sub.1 is dimensioned to fit in a high
pressure freezing unit for high pressure freezing of the specimen
holding element (100, 100').
3. The modular specimen holder (10, 10') according to claim 1,
wherein the second distance d.sub.2 is dimensioned to fit in an
X-ray crystallography unit for conducting an X-ray crystallography
of the specimen.
4. The modular specimen holder (10, 10') according to claim 1,
further comprising a cartridge (300) connectable with the specimen
holding element (100, 100') and separable from the specimen holding
element (100, 100'); the cartridge (300) comprising an
encapsulating element (320) in its interior, wherein: the tubule
(120, 120') and the encapsulating element (320) are formed such
that the tubule (120, 120') can be placed inside the encapsulating
element (320).
5. The modular specimen holder (10, 10') according to claim 4,
wherein the encapsulating element (320) is constructed as a cavity
inside the cartridge.
6. The modular specimen holder (10, 10') according to claim 4,
wherein the cartridge (300) is constructed as a single piece.
7. The modular specimen holder (10, 10') according to claim 4,
wherein the cartridge (300) comprises connection means (330) to
connect the cartridge (300) with the base element (110, 110') of
the specimen holding element.
8. The modular specimen holder (10, 10') according to claim 4,
wherein the specimen holding element (100, 100') is designed to fit
into the cartridge (300).
9. The modular specimen holder (10, 10') according to claim 1,
wherein at least a part of the tubule (120, 120') is made of
polyimide.
10. The modular specimen holder (10, 10') according to claim 1,
wherein the tubule (120, 120') comprises a pin (121, 121') and a
holding element (122, 122'), wherein the pin (121, 121') is made of
a metal material and wherein the holding element (122, 122') is
adapted to hold the specimen and is made of polyimide.
11. The modular specimen holder (10, 10') according to claim 1,
wherein the base element (110, 110') is of cylindrical or
essentially cylindrical shape comprising a lateral area (111, 111')
and a top (112, 112') perpendicular to the lateral area (111,
111').
12. The modular specimen holder (10, 10') according to claim 11,
the top (112, 112') of the base element (110, 110') being
constructed as a plane or essentially plane sheet or as a plane or
essentially plane sheet with a stepped outer rim (115, 115').
13. The modular specimen holder (10, 10') according to claim 1, the
base element (110, 110') being hollow in its interior and the
bottom of the base element comprising or forming an opening.
14. The modular specimen holder (10, 10') according to claim 1,
wherein the extension element (200, 200') comprises connection
means (202) adapted to interact with the base element (110, 110'),
thus establishing the connection of the specimen holding element
(100, 100') and the extension element (200, 200').
15. The modular specimen holder (10, 10') according to claim 13,
wherein the extension element (200, 200') comprises connection
means (202) adapted to interact with the base element (110, 110'),
thus establishing the connection of the specimen holding element
(100, 100') and the extension element (200, 200'), the connection
means (202, 202') of the extension element being adapted to be
inserted into the interior of the base element (110, 110') through
the opening in the bottom of the base element (110, 110').
16. The modular specimen holder (10, 10') according to claim 14,
wherein the connection means (202, 202') are adapted to establish a
magnetic and/or mechanical connection of the specimen holding
element (100, 100') and the extension element (200, 200').
17. The modular specimen holder (10, 10') according to claim 1,
wherein a diameter (d.sub.3) of the bottom of the extension element
(200, 200') is essentially 12 mm.
18. The modular specimen holder (10, 10') according to claim 1,
wherein the first distance d.sub.1 from the bottom of the base
element (110, 110') to the top of the tubule (120, 120') is in a
range between 15 mm and 19 mm.
19. The modular specimen holder (10, 10') according to claim 1,
wherein the second distance d.sub.2 from the bottom of the
extension element (200, 200') to the top of the tubule (120, 120')
is in a range of 22 mm.+-.1.5 mm.
20. The modular specimen holder (10, 10') according to claim 1,
wherein the first distance d.sub.1 from the bottom of the base
element (110, 110') to the top of the tubule (120, 120') is
dimensioned to fit in a high pressure freezing unit.
21. The modular specimen holder (10, 10') according to claim 1,
wherein the second distance d.sub.2 from the bottom of the
extension element (200, 200') to the top of the tubule (120, 120')
is dimensioned according to the SPINE standard.
22. A method of high pressure freezing and X-ray crystallography of
a specimen using a modular specimen holder (10, 10') according to
claim 4, comprising the steps of: connecting the specimen holding
element (100, 100') holding the specimen and the cartridge (300)
with each other; placing the connected specimen holding element
(100, 100') and the cartridge (300) in a high pressure freezing
unit; conducting a high pressure freezing of the specimen by means
of the high pressure freezing unit; separating the frozen specimen
holding element (100, 100') and the cartridge (300) from each
other; connecting the frozen specimen holding element (100, 100')
with the extension element (200, 200'); placing the connected
specimen holding element (100, 100') and the extension element
(200, 200') in an X-ray crystallography unit; and conducting an
X-ray crystallography of the frozen specimen by means of the X-ray
crystallography unit.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is the U.S. national phase of
International Application No. PCT/EP2017/067696 filed Jul. 13,
2017, which claims priority of European Application No. 16186050.7
filed Aug. 26, 2016, the entire disclosure of which is incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to modular specimen holders
for high pressure freezing and X-ray crystallography and to a
method of high pressure freezing and X-ray crystallography of a
specimen using a modular specimen holder of that kind.
BACKGROUND OF THE INVENTION
[0003] X-ray crystallography is a method of identifying the atomic
and/or molecular structure of a crystal. The specimen is targeted
by a beam of X-ray radiation, which is diffracted by the
crystalline structure of the specimen. By measuring the angles and
intensities of the diffracted beams, a three-dimensional model of
the density of electrons within the crystal can be produced. From
this electron density, the mean positions of the atoms in the
crystal can be determined, as well as their chemical bonds, their
disorder and various other information. The X-ray radiation for the
X-ray crystallography can e.g. be produced by a synchrotron.
[0004] X-ray crystallography can be used to determine the structure
of large biomolecules such as proteins. In order to reduce
X-ray-induced radiation damages to the protein crystals during the
X-ray crystallography, the specimen can be cooled to cryogenic
temperatures prior to the X-ray crystallography by means of high
pressure freezing.
[0005] High pressure freezing (HPF) is a method for rapid freezing
of water-containing specimen or preparations under high pressure.
By means of the high pressure freezing a vitrification (i.e.,
freezing with no formation of ice crystals) of the specimen is
achieved, in the course of which water is transformed from a liquid
to an amorphous state without inducing the nucleation of ice
crystals. Thus, a cryo-fixation or cryo-immobilisation of the
specimen can be achieved. Cellular constituents of the specimen can
be fixed without introducing significant structural
alterations.
[0006] For an elaborate description of high pressure freezing of
protein crystals it is referred to Burkhardt, Anja et al. "Fast
High-Pressure Freezing of Protein Crystals in Their Mother Liquor."
Acta Crystallographica Section F: Structural Biology and
Crystallization Communications 68.Pt 4 (2012): 495-500. PMC. Web.
24 Aug. 2016.
[0007] For high pressure freezing of a specimen, usually a first
specimen holder is used. After the freezing process, the specimen
has to be separated from the first specimen holder and has to be
placed on a second specimen holder for X-ray crystallography. This
transport between different specimen holders is an elaborate and
time consuming procedure and yields the danger of damaging and
contaminating the specimen.
[0008] It is desirable to minimise the transport efforts and to
reduce the danger of damaging or contaminating the specimen between
the steps of high pressure freezing and X-ray crystallography.
SUMMARY OF THE INVENTION
[0009] This object is achieved by modular specimen holders and by a
method of high pressure freezing and/or X-ray crystallography of a
specimen according to the features of the independent claims.
Further advantages and embodiments of the invention will become
apparent from the dependent claims and the following description
and the embodiments according to the appended figures. Advantages
and embodiments of the modular specimen holders according to the
invention and of the method according to the invention arise from
the following in an analogous manner.
[0010] The modular specimen holders according to the invention are
particularly suitable for specimen comprising or consisting of
biomolecules, particularly protein crystals, whose crystal
structure shall be analysed in the course of the X-ray
crystallography. The X-ray radiation for the X-ray crystallography
can especially be produced by a synchrotron.
[0011] According to a first aspect of the invention, the modular
specimen holder comprises a specimen holding element and an
extension element, which are connectable with each other and, vice
versa, separable from each other.
[0012] The specimen holding element comprises a tubule and a base
element. The tubule is adapted to hold the specimen, particularly
at a top of the tubule. For this purpose, the tubule can comprise a
corresponding holding element, e.g. a loop. The base element is
adapted to hold the tubule, particularly at the bottom of the
tubule, i.e. at that tubule end, which does not hold the specimen.
The tubule can particularly be firmly attached to the base element
or be detachable from the base element. The base element
particularly comprises a corresponding holding element for the
tubule, which can e.g. be arranged on a top of the base element.
The tubule is particularly arranged perpendicular or essentially
perpendicular to a top surface of the base element.
[0013] A first distance from a bottom of the base element to the
top of the tubule is d.sub.1. This first distance d.sub.1 is
particularly dimensioned to fit in a high pressure freezing unit
for high pressure freezing of the specimen holding element. The
specimen holding element is thus specifically designed and
dimensioned for a specific type of high pressure freezing unit. In
order to conduct high pressure freezing of the specimen, preferably
only the specimen holding element can be used without being
connected to the extension element.
[0014] However, the specimen holding element per se can usually not
be used for X-ray crystallography units, since the specimen holding
element does not fit into corresponding crystallography units.
Usually the specimen holding element is too small for these units.
Thus, for conducting an X-ray crystallography of the specimen, the
specimen holding element and the extension element are connected
with each other. To this end, the extension element is adapted to
be connected to the base element, particularly to a bottom surface
of the base element. When the extension element and the base
element are connected with each other, a second distance from a
bottom of the extension element to the top of the tubule is
d.sub.2. The second distance d.sub.2 is larger than the first
distance d.sub.1. The second distance d.sub.2 is particularly
dimensioned to fit in an X-ray crystallography unit for conducting
an X-ray crystallography of the specimen.
[0015] The thus assembled modular specimen holder can be
specifically designed and dimensioned for a specific type of X-ray
crystallography unit. Particularly, the extension element is
specifically designed and dimensioned to adapt the specimen holding
element, which is as such not usable for X-ray crystallography,
into a specimen holder usable in the corresponding X-ray
crystallography unit.
[0016] According to a second aspect of the invention, the modular
specimen holder comprises a specimen holding element and a
cartridge, which are connectable with each other and, vice versa,
separable from each other.
[0017] Analogously to the above description, this specimen holding
element comprises a tubule adapted to hold the specimen and a base
element adapted to hold the tubule.
[0018] The cartridge comprises an encapsulating element in its
interior. The tubule and the encapsulating element are formed such
that the tubule can be placed inside the encapsulating element.
This encapsulating element can especially be constructed as a
cavity inside the cartridge, particularly as a tubular cavity. The
specimen holding element is thus designed to fit into the
cartridge.
[0019] A diameter of said encapsulating element is particularly
only slightly larger than a diameter of the tubule. After the
freezing process, the connected specimen holding element and
cartridge can be stored in the storage area. For X-ray
crystallography, the specimen holding element is separated from the
cartridge.
[0020] The specimen holding element is thus especially designed and
dimensioned to perfectly fit into the cartridge. The combination of
specimen holding element and cartridge is specifically designed for
a specific type of high pressure freezing unit. In order to conduct
high pressure freezing of the specimen, the specimen holding
element and the cartridge are connected with each other.
[0021] The invention enables an easy way to conduct both the high
pressure freezing and the subsequent X-ray crystallography of a
specimen with the same specimen holder. In contrast to the
invention, according to the prior art, the specimen is first frozen
using a first specimen holder, which can only be used in the
corresponding high pressure freezing unit; then, the specimen has
to be separated from the first specimen holder and has to be placed
onto a second specimen holder, which can only be used in the
corresponding X-ray crystallography unit. This second specimen
holder can be inserted into the crystallography unit and X-ray
crystallography can be conducted. In contrast to that, according to
the present invention, the specimen does not have to be transported
between different specimen holders, which is an elaborate and time
consuming procedure and yields the danger of damaging or
contaminating the specimen.
[0022] The invention also relates to a (first) system comprising
the modular specimen holder according to the first aspect of the
invention and an X-ray crystallography unit for conducting an X-ray
crystallography of the specimen. The invention also relates to a
(second) system comprising the modular specimen holder according to
the second aspect of the invention and a high pressure freezing
unit for high pressure freezing of the specimen. Finally, the
invention also relates to a combined system comprising the first
and second systems.
[0023] In the course of the method according to the present
invention, the specimen holding element holding the specimen is
connected with the cartridge. The connected cartridge and specimen
holding element are placed in the corresponding high pressure
freezing unit. High pressure freezing of the specimen is conducted
by means of the high pressure freezing unit. Afterwards, the frozen
specimen can either immediately be analysed in the course of the
crystallography. Alternatively, the frozen specimen can be stored
in an expedient cryo-storage area, which can e.g. be cooled by
liquid nitrogen to cryogenic temperatures. For this purpose, the
connected specimen holding element and cartridge can be transported
to and stored in the corresponding storage area. From there, the
specimen holding element is collected and transported to the
crystallography unit.
[0024] In order to conduct the X-ray crystallography, the frozen
specimen holding element is separated from the cartridge and
connected with the extension element. The connected specimen
holding element and extension element forming the modular specimen
holder are placed in the corresponding X-ray crystallography unit
and the X-ray crystallography of the frozen specimen is conducted
by means of the X-ray crystallography unit.
[0025] The extension element can be connected with the specimen
holding element straight after the freezing and the combined
specimen holding element, cartridge and extension element can be
stored in a cryogenic storage area. Alternatively, the connected
specimen holding element and cartridge can be stored.
Alternatively, only the specimen holding element can be stored in
the storage area and can be connected with the extension element
after having been collected from the storage area.
[0026] A corresponding high pressure freezing unit is manufactured
by the applicant under the trade name of Leica EM ICE, which
replaces a former model named Leica EM HPM. According to a
particularly advantageous embodiment of the invention, the distance
from the bottom of the base element to the top of the tubule is
dimensioned to fit in the high pressure freezing unit designed in
the way of a Leica EM ICE. In this Leica EM ICE device or a device
designed like that, liquid nitrogen is expediently used as both a
pressure transfer medium and a coolant. The freezing cycle in this
system can expediently be conducted as follows:
[0027] The freezing process is conducted inside a chamber, inside
which the specimen is placed. Pressure build up is conducted
downstream a closed vent, which is located in front of a chamber.
By opening this vent nitrogen is injected into the chamber. This
nitrogen stream is restricted by means of a nozzle at an exit of
the chamber. Hence, pressure is built up inside the chamber. This
stream of nitrogen usually does not have cryogenic temperatures.
Freezing of the specimen is conducted after this pressure build up,
i.e. after the specimen is set under pressure. For this purpose a
compressed cryogenic medium with cryogenic temperature, e.g. liquid
nitrogen, is conducted into the chamber.
[0028] By this device a pressure rise of especially up to 2000 bar
in less than 10 ms can be achieved. The sample can be cooled
immediately after reaching the 2000 bar pressure at a cooling rate
of typically 5000 K/sec or higher. For a more detailed description
of high pressure freezing it is referred to the documents DE 1 806
741 A1, DE 100 15 773 A1, or DE 100 65 143 A1.
[0029] The high pressure freezing unit Leica EM ICE or a
corresponding unit designed in that way comprises a loading
station. In order to conduct the freezing process, first the
specifically constructed cartridge is placed in this loading
station. Afterwards, the specimen holding element is placed in the
loading station and is inserted into the cartridge. By closing a
cover or lid of the loading station, the cartridge and the specimen
holding element are connected with each other and the high freezing
process is automatically triggered. After the freezing process, the
combination of cartridge and specimen holding element is
automatically ejected by the Leica EM ICE or the corresponding unit
into a cooling bath of a cryogenic medium, especially liquid
nitrogen, e.g. in a Dewar vessel.
[0030] This cartridge and the specimen holding element are
specifically designed and dimensioned for the Leica EM ICE or for
high pressure freezing unit designed in that way to enable an
optimal freezing of the specimen. The specimen holding element is
especially designed and dimensioned to perfectly fit into the
cartridge. After the freezing process, the connected specimen
holding element and cartridge can be stored in the storage area.
For the X-ray crystallography, the specimen holding element is
separated from the cartridge.
[0031] Advantageously, the distance from the bottom of the base
element to the top of the tubule is in the range between 15 mm and
19 mm. In this case, the specimen holding element is designed to
precisely fit into the cartridge and into the Leica EM ICE or a
device like that. The Leica EM ICE allows a maximum height of 19 mm
of the corresponding specimen holding element.
[0032] However, as pointed out before, the specimen holding element
is per se usually not suitable for X-ray crystallography units.
Sample holders for X-ray crystallography units are usually
constructed by various manufacturers according to standards of the
so called SPINE consortium. SPINE (Structural Proteomics IN Europe)
was founded to push forward with technologies aimed at biomedically
relevant targets and to generate a pan-European integration on
biomedically focused structural proteomics. For a detailed
explanation of SPINE see Stuart, D. I., Jones, E. Y., Wilson, K. S.
& Daenke, S. (2006), Acta Cryst. D62, -2--1.
[0033] The specific characteristics of corresponding sample holders
are given in the document "SPINE SAMPLE HOLDER & VIAL
SPECIFICATIONS-L-R05" revised on 15 Apr. 2014, which is available
on the homepage: [0034] https://www.embl.fr/spinesampleholder/
According to this SPINE standard, a corresponding sample holder
consists of a cap and a pin. A crystal support, especially a loop,
is mounted on said pin. Said cap is a support of the pin. The
sample holder length, which is defined as the distance from the
base of the cap to the crystal (or beam position), e.g. the top of
the pin, is 22 mm. By this fixed sample holder length the SPINE
consortium wants to achieve a compatibility of different kinds of
sample changers for X-ray crystallography units. Thus, the distance
from the bottom of the cap, at which a sample changer grabs the
sample holder or is attached to the sample holder, to the position
of the sample, at which an X-ray beam hast to be directed in order
to conduct the crystallography, is always 22 mm.
[0035] Advantageously, when the extension element and the base
element are connected with each other, the second distance d.sub.2
from the bottom of the extension element to the top of the tubule
is in the range of 22 mm.+-.1.5 mm. Since the specimen is not
necessarily placed precisely at the top of the tubule, there is
preferably a tolerance range of .+-.1.5 mm. The distance from the
bottom of the extension element to the top of the tubule is thus
preferably dimensioned according to the SPINE standard. The modular
sample holder can thus be used in any kind of X-ray crystallography
unit designed according to the SPINE standard. Preferably, a
diameter of the bottom of the extension element is essentially or
precisely 12 mm. This diameter is also set by the SPINE
standard.
[0036] The invention therefore particularly provides a modular
specimen holder, which can be used for high pressure freezing by
means of a Leica EM ICE or by a unit designed that way and which is
compatible with the SPINE standard and can thus be used for X-ray
crystallography by means of an X-ray crystallography unit designed
according to the SPINE standard.
[0037] Preferably, the cartridge is constructed as a single piece.
The cartridge is thus especially constructed as a solid element
which is not separable. However, the cartridge could also be
embodied as two or several shells separable from each other.
[0038] Advantageously, the cartridge comprises connection means to
connect the cartridge with the base element of the specimen holding
element. Preferably, these connection means are constructed as
magnetic connection means. These magnetic connection means
particularly interact with the base element made of a metal
material and thus establish a force-fitted connection between
cartridge and specimen holding element.
[0039] Preferably, at least a part of the tubule is made of
polyimide, especially Kapton. According to a particularly
advantageous embodiment the tubule comprises a pin made of a metal
material and a holding element adapted to hold the specimen made of
polyimide, especially Kapton. Protein crystals are especially
dissolved in a specific solution. The crystals can be placed in the
Kapton-made element, particularly in the holding element, as
specimen in this solution. The base element is especially made of
steel anticorrosion coated or ferromagnetic stainless steel
(especially stainless steel 430F or equivalent).
[0040] Advantageously, the base element is of cylindrical or
essentially cylindrical shape comprising a lateral area and a top
perpendicular to the lateral area. Preferably, the top of the base
element is constructed as a plane or essentially plane sheet. This
sheet especially can e.g. comprise a hole, into which the tubule
can be inserted. This hole is especially located in the centre of
the plane sheet. The top can preferably be constructed as a plane
or essentially plane sheet with a stepped outer rim, particularly
in order to place the specimen holding element precisely inside the
cartridge.
[0041] The base element is preferably hollow in its interior. The
bottom of the base element preferably comprises an opening.
Particularly, essentially the entire bottom of the base element can
form this opening. In the hollow interior of the base element
expedient guiding means for a precise insertion of the tubule into
the corresponding tube of the cartridge can be arranged.
Particularly, a mechanical guiding element can be attached to these
guiding means. For this purpose, the mechanical guiding element can
especially be inserted into the specimen holding element through
the opening in the bottom of the base element. By means of this
mechanical guiding element, the specimen holder can especially be
inserted in the cartridge.
[0042] Preferably, the extension element comprises connection means
adapted to interact with the interior of the base element,
especially with its inner surfaces, thus establishing the
connection of the specimen holding element and the extension
element. In the interior of the base element, corresponding second
connection means can be arranged, which can interact with the
extension element's connection means. The connection means of the
extension element are advantageously adapted to be inserted into
the interior of the base element through the opening in the bottom
or formed by the bottom of the base element.
[0043] The corresponding connection means are advantageously
adapted to establish a magnetic and/or mechanical connection of the
specimen holding element and the extension element. The connection
means of the extension element can for example be constructed as
one or several clamping elements, establishing a force-fitted
connection between specimen holding element and extension element.
In this case, there are not necessarily second connection means in
the interior of the base element. The force-fitted connection can
be established by the clamping means pressing or forcing or being
biased against the interior wall of the base element. A
force-fitted connection can also be established by magnetic
connection means which can interact with metal interior wall
elements of the base element. The connection means can especially
establish a form-fitted connection. For this purpose, the
connection means and/or the second connection means can be embodied
as threads.
[0044] It should be noted that the previously mentioned features
and the features to be further described in the following are
usable not only in the respectively indicated combination, but also
in further combinations or taken alone, without departing from the
scope of the present invention.
BRIEF DESCRIPTION OF THE DRAWING VIEWS
[0045] The present invention will now be described further, by way
of example, with reference to the accompanying drawings, in
which
[0046] FIG. 1 schematically shows a preferred embodiment of a
modular specimen holder with specimen holding element and extension
element according to the invention in different perspective
views,
[0047] FIG. 2 schematically shows a preferred embodiment of a
modular specimen holder with specimen holding element and extension
element according to the invention in different perspective
views,
[0048] FIG. 3 schematically shows a preferred embodiment of a
modular specimen holder with specimen holding element, extension
element, and cartridge according to the invention in different
perspective views, and
[0049] FIG. 4 schematically shows a part of a high pressure
freezing unit with a specimen holding element and a cartridge of a
modular specimen holder according to the invention in a perspective
view.
DETAILED DESCRIPTION
[0050] In FIG. 1 a preferred embodiment of a modular specimen
holder with specimen holding element and extension element
according to the invention is schematically shown and labelled as
10. The modular specimen holder 10 is shown in FIG. 1a in a
perspective exploded view and in FIG. 1b in its compound state in a
perspective view. Identical reference signs in the figures refer to
identical or structurally identical elements.
[0051] In FIG. 1a, the two modules of the specimen holder 10, i.e.
the specimen holding element 100 and the extension element 200 are
shown separated from each other.
[0052] The specimen holding element 100 comprises a base element
110 and a tubule 120. The tubule 120 comprises a pin 121 made of a
metal material on which a holding element 122 made of polyimide,
preferably Kapton, is mounted. A specimen can be placed in this
holding element 122. The holding element 122 is particularly
constructed as a small Kapton tube, inside which protein crystals
can be placed as specimen in a specific solution, in which they are
dissolved.
[0053] The holding element 122 can e.g. also be constructed as a
loop. The tubule 120 can especially be constructed as a so called
Hampton pin.
[0054] The base element 110 is adapted to hold the tubule 120 and
is of cylindrical or essentially cylindrical shape comprising a
lateral area 111 and a top 112 perpendicular to the lateral area
111. The interior of the base element 110 is particularly hollow.
Guiding means can be provided on the inner wall of the lateral area
111 in order to insert the specimen holding element 100 in a
cartridge as will be explained with reference to FIG. 3 and FIG.
4.
[0055] The top 112 can be constructed as a plane or essentially
plane sheet, particularly with a stepped outer rim 115. This
stepped outer rim 115 is especially provided in order to enable a
centred positioning of the specimen holding element 100 inside the
cartridge, as will be explained with reference to FIG. 3.
[0056] There is no sheet at the bottom of the base element 110.
Thus, the bottom of the base element 110 forms an opening. The base
element 110, i.e. the lateral area 111 and the top 112, are made of
metal, e.g. ferromagnetic stainless steel.
[0057] The base element 110 particularly comprises a holding
element for the tubule 120 in form of a borehole 113 in the centre
of the sheet 113, into which the tubule 120 can be inserted. The
tubule 120 is thus arranged perpendicular or essentially
perpendicular to the top 112 of the base element.
[0058] There are several more boreholes 114 circumferential to the
centre of the sheet 112 for conducting liquid nitrogen in the
course of the high pressure freezing.
[0059] The extension element 200 comprises a base 201 on which
connection means 202 are arranged. These connection means 202 are
adapted to connect the specimen holding element 100 with the
extension element 200.
[0060] The connection means 202 can be inserted into the hollow
interior of the base element 110 through the opening in the bottom
of the base element 110. In this particular example, the connection
means 202 are embodied as clamping elements, establishing a
force-fitted connection between specimen holding element 100 and
extension element 200 by pressing or forcing against the interior
wall of the base element 110.
[0061] The modular specimen holder 10 is shown in FIG. 1b in its
compound state, i.e. with the specimen holding element 100 and the
extension element 200 connected with each other. In FIG. 1b, a
distance from a bottom of the extension element 200 to the top of
the tubule 120 is d.sub.2. This distance d.sub.2 is 22 mm.+-.1.5 mm
and is hence dimensioned according to the SPINE standard.
Particularly, the set distance of 22 mm of the SPINE standard
refers to the distance from the bottom of the extension element 200
to the specimen. Thus, if the specimen is not placed exactly at the
edge of the holding element 122, the distance d.sub.2 can be
slightly larger than 22 mm.
[0062] Thus, the distance d.sub.2 is dimensioned to fit in an X-ray
crystallography unit for conducting an X-ray crystallography of the
specimen constructed according to the SPINE standard. Moreover, a
diameter d.sub.3 of a bottom of the extension element 200 is
essentially or precisely 12 mm and thus also dimensioned according
to the SPINE standard.
[0063] In its compound state, the modular specimen holder 10 thus
fulfils the requirements of the SPINE standard. However, the
compound modular specimen holder 10 cannot be used for specific
types of high pressure freezing units, particularly Leica EM ICE or
of similar design.
[0064] When separated from each other, however, the specimen holder
can be used in the Leica EM ICE or a device designed in that way.
This device allows a maximum height of 19 mm of the corresponding
specimen holding element. Thus, a distance d.sub.1 from a bottom of
the base element 110 to the top of the tubule 120 is at most 19 mm,
particularly in the range between 15 mm and 19 mm, and is thus
dimensioned to fit in a high pressure freezing unit in form of the
Leica EM ICE.
[0065] In FIG. 2 another preferred embodiment of a modular specimen
holder according to the invention is schematically shown and
labelled as 10' with specimen holding element 100' and extension
element 200'.
[0066] FIG. 2a shows the modular specimen holder 10' in a
perspective sectional view with specimen holding element 100' and
extension element 200' separated from each other. FIG. 2b is a
perspective a sectional view of specimen holding element 100' and
extension element 200' connected with each other. FIG. 2c is a
perspective view analogously to FIG. 2b.
[0067] Analogously, to the modular specimen holder 10 of FIG. 1,
also the modular specimen holder 10' of FIG. 2 comprises a base
element 110' adapted to hold the tubule 120' comprising a metal pin
121' and a Kapton holding element 122'. A holding element 113' for
the tubule 120' is constructed as a cylinder.
[0068] The base element 110' is of cylindrical or essentially
cylindrical shape. In contrast to the base element 110 of FIG. 1,
this base element 110' is not hollow in its interior but an
essentially solid cylinder with lateral area 111' and top 112'.
Boreholes 114' inside the base element 110' are provided for
conducting liquid nitrogen in the course of the high pressure
freezing. The cylindrical base element 110' can in this case have a
height from the bottom to the top surface 112' in the range between
e.g. 1 mm and 3 mm.
[0069] The extension element 200' comprises a base 201' and
connection means 202'. In this example the connection means 202 are
constructed as magnetic connection means in order to establish a
force-fitted connection between specimen holding element 100' and
extension element 200'. For this purpose one or several magnets
202' are provided which can interact with the base element 110'
made of metal.
[0070] Analogously to FIG. 1b, the first distance d.sub.1 from the
bottom of the base element 110' to the top of the tubule 120' is in
the range between 15 mm and 19 mm, and is thus dimensioned to fit
in a high pressure freezing unit in form of the Leica EM ICE.
[0071] The second distance from the bottom of the extension element
200' to the top of the tubule 120' is d.sub.2 and is in the range
22 mm.+-.1.5 mm and is hence dimensioned according to the SPINE
standard.
[0072] Moreover, the diameter d.sub.3 of the bottom of the
extension element 200' is essentially or precisely 12 mm and thus
also dimensioned according to the SPINE standard.
[0073] According to the invention, the specimen holding element is
also connectable with a cartridge for high pressure freezing, as
will now be explained in reference to FIG. 3.
[0074] FIG. 3 shows the specimen holding element 100' and the
extension element 200' of FIG. 2 as well as a corresponding
cartridge 300.
[0075] FIG. 3a shows the three modules 100', 200', and 300
separated from each other in a perspective sectional view. These
modules 100', 200', and 300 are shown connected with each other in
a perspective sectional view in FIG. 3b and in a perspective view
in FIG. 3c
[0076] The cartridge 300 has an encapsulating element 320 in its
interior in the form of a tubular cavity. This tubular cavity 320
is located inside a casing 310 of the cartridge 300. The tubule
120' is adapted to be placed inside this tubular cavity 320. A
diameter of said tubular cavity 320 is particularly only slightly
larger than a diameter of the tubule 120'.
[0077] Moreover, connection means 330 are provided inside the
casing 310 in order to connect the cartridge 300 with the specimen
holding element 100'. These connection means 330 are preferably
constructed as magnetic connection means 330, e.g. one or several
magnets. These magnets 330 can interact with the base element 110'
in order to establish a force-fitted connection between cartridge
300 and the metal base element 110'.
[0078] In FIG. 4, a part of a high pressure freezing unit in form
of the Leica EM ICE is schematically shown in a perspective view.
Particularly, a loading station 400 of the Leica EM ICE is shown,
into which the specimen is loaded for high pressure freezing.
[0079] The loading station 400 comprises a chamber 401, into which
firstly the cartridge 300 is loaded by a mechanical guiding element
402, e.g. a rod. Afterwards the specimen holding element 100' is
positioned in the chamber by the mechanical guiding element 402 and
is thus inserted into the cartridge 300.
[0080] For loading the specimen holding element 100' into the
chamber 401, the mechanical guiding element 402 is connected with
guiding means in the interior of the base element 110' of the
specimen holding element 100'. The mechanical guiding element 402
enables precise insertion of the specimen holding element 100' in
the cartridge 300 without damaging the specimen.
[0081] It is also possible to provide two different mechanical
guiding elements; a first one for loading the cartridge into the
chamber 401 and a second one for the specimen holder.
[0082] When both the cartridge 300 and the specimen holding element
100' are loaded into the chamber 401, a lid 404 with a handle 404
is closed. Closing this lid 403 triggers the high pressure freezing
of the specimen. For this purpose, the cartridge 300 and the
specimen holding element 100' are connected with each other and
transported through a conduit 405 into the interior of the Leica EM
ICE, where the high pressure freezing process is conducted.
[0083] After the freezing process, the combination of cartridge 300
and specimen holding element 100' is automatically ejected by the
Leica EM ICE into a cooling bath of a cryogenic medium, especially
liquid nitrogen, e.g. in a Dewar vessel.
REFERENCE SIGNS
[0084] 10 modular specimen holder [0085] 100 specimen holding
element [0086] 110 base element [0087] 111 lateral area of the base
element [0088] 112 top of the base element, sheet [0089] 113
holding element for the tubule 120, borehole [0090] 114 boreholes
[0091] 120 tubule [0092] 121 pin [0093] 122 holding element [0094]
200 extension element [0095] 201 base [0096] 202 connection means,
clamping elements [0097] 10' modular specimen holder [0098] 100'
specimen holding element [0099] 110' base element [0100] 111'
lateral area of the base element [0101] 112' top of the base
element, sheet [0102] 113' holding element for the tubule 120'
[0103] 114' boreholes [0104] 120' tubule [0105] 121' pin [0106]
122' holding element [0107] 200' extension element [0108] 201' base
[0109] 202' connection means, magnet [0110] 300 cartridge [0111]
310 casing of the cartridge [0112] 320 encapsulating element,
tubular cavity [0113] 330 connection means, magnets [0114] 400
loading station of a high pressure freezing unit [0115] 401 chamber
[0116] 402 mechanical guiding element, rod [0117] 403 lid [0118]
404 handle [0119] 405 conduit
* * * * *
References